Controversy exists as to the proton stoichoimetries of the individual steps in oxidative phosphorylation. Consequently, once widely accepted ATP/O and ATP/site stoichiometries are in doubt. The uncertainties are large, and various proposals differ by 100% or more for individual ATP/site ratios. The major objectvie of this project is to evaluate quantitatively the ATP/O and ATP/site ratios of heart mitochondria and submitochondrial particles (SMP) from unequivocal steady state determinations of products and ractants using the theorites of equilibrium and nonequilibrium thermodynamics. This approach avoids the difficulties of transient H-+/e--measurements and the uncertain corrections of Delta approximately mu H+ determinations for nonspecific binding of permeant probe ions. Upper and lower limits to the ATP stoichiometries will be evaluated as follows: 1) Upper limits to ATP/2e- individually at Site 1, Site 2 and Site 3 will be determined in mitochondria from Delta G-R/delta G-P measured at static head (state 4) where Delta G-R is the redox free energy change across each site and Delta G-P is the free energy change of ATP synthesis. 2) Lower limits for ATP/2e- at Site 2 and Site 2 wil be determined from Delta G-R/Delta G-/P during ATP-dirven reverse electron transfer. 3) Upper limits to ATP/O in SMP oxidzing succinate or NADH at static head will be determined from Delta G-R/Delta G-P. 4) Lower limits to ATP/O in mitochondria oxidizing a variet of NAD and FAD-linked respiratory substrates will be determined from ADP-induced oxygen jumps. 5) Using the theory of linear nonequilibrium thermodynamics, each limit will be corrected for the degree of coupling, q, to yield z, the phenomenological stoichiometry, an estimate of the mechanistic stoichiometry. The proposed studies will place narrow empirical limits to the mechanistic ATP stoichiometries of oxidative phosphorylation by heart mitochondria consistent with a single scheme of chemiosmotic coupling.